I propose to investigate the biochemical and molecular biological mechanisms of phorbol diester induced differentiation of human myeloid leukemia cells. Activation of the Ca2+/phospholipid dependent protein kinase, termed protein kinase C, is the likely membrane transduction signal by which the phorbol diesters and mononuclear cell conditioned media induce macrophage maturation of human myeloid leukemia cells. Protein kinase C from human promyelocytic leukemia cells, HL-60, will be purified to homogeneity using classical and affinity purification techniques. Defined diacylglycerols will be prepared and their activities as kinase activators, ligands for the phorbol diester receptor and inducers of maturation will be compared in purified enzyme preparations as well as in whole cells. Phorbol diester induced phospoprotein alterations will be characterized in whole cells and in nuclei. The effects of phorbol diester induced HL-60 maturation on the activity of several oncogenes, including myc and N-ras, will be determined by examining RNA levels and nuclear transcription. Once the level of oncogenic control has been ascertained, precise molecular mechanisms of control will be postulated and tested. The long term objective is to understand leukemic as well as normal myeloid maturation and its relationship to oncogene activity and control. The major scientific disciplines involved are Hematology, Biochemistry and Molecular Biology. The intense study of in vitro leukemic cell maturation will define the precise molecular defect which leads to the leukemic state and understanding the mechanisms of differentiation will aid in finding agents active as inducers of cell differntiation for future clinical application.